Limits...
Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

Ghosh S, Salsbury FR, Horita DA, Gmeiner WH - Nucleic Acids Res. (2011)

Bottom Line: Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect.DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+).A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Program in Molecular Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

ABSTRACT
We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

Show MeSH

Related in: MedlinePlus

Structure of the DNA hairpins investigated in the present studies. Each of the hairpins includes a 10 bp stem capped by the pentanucleotide CGAAG to promote intramolecular hairpin formation. The control sequence A10–T10 consists of all native nucleotides. The three FdU-containing sequences differ in the location of FdU nucleotides as shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105383&req=5

Figure 2: Structure of the DNA hairpins investigated in the present studies. Each of the hairpins includes a 10 bp stem capped by the pentanucleotide CGAAG to promote intramolecular hairpin formation. The control sequence A10–T10 consists of all native nucleotides. The three FdU-containing sequences differ in the location of FdU nucleotides as shown.

Mentions: The DNA sequences used for these studies were designed to favor intramolecular hairpin formation and to be stable at physiological temperature. Each of the four hairpin DNA sequences consisted of a 10 bp stem consisting of all A–T (or A–FdU) base pairs. The loop region included the GAA sequence that was previously shown to promote hairpin formation (12). The loop was closed by a single C–G base pair. Gel-electrophoresis studies (15% native PAGE gel) demonstrated a single species was present (Supplementary Figure S8) and subsequent 1H NMR studies demonstrated the chemical shift characteristic for the C–G base pair closing the loop in a similar sequence (13). Four DNA hairpins were designed (Figure 2). The sequence ‘A10–T10’ included 10 consecutive T nucleotides at the 5′-terminus and the corresponding 10 consecutive A nucleotides at the 3′-terminus. The ‘5′-FdU’ sequence was identical to the A10–T10 sequence except all of the T nucleotides were substituted by FdU. The ‘3′-FdU’ hairpin DNA sequence had the location of the 10 consecutive A and FdU nucleotides reversed relative to the 5′-FdU hairpin with the 10 consecutive FdU nucleotides located at the 3′-terminus. The ‘Alt-FdU’ sequence had alternating A and FdU nucleotides throughout the stem and thus lacked consecutive FdU nucleotides. These DNA hairpin sequences were designed to evaluate the structural requirements for FdU substitution on formation of complexes with divalent metal ions, including Zn2+ and Mg2+. All DNA sequences were prepared at the University of Calgary DNA Core laboratory and purified by gel-filtration chromatography.Figure 2.


Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

Ghosh S, Salsbury FR, Horita DA, Gmeiner WH - Nucleic Acids Res. (2011)

Structure of the DNA hairpins investigated in the present studies. Each of the hairpins includes a 10 bp stem capped by the pentanucleotide CGAAG to promote intramolecular hairpin formation. The control sequence A10–T10 consists of all native nucleotides. The three FdU-containing sequences differ in the location of FdU nucleotides as shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105383&req=5

Figure 2: Structure of the DNA hairpins investigated in the present studies. Each of the hairpins includes a 10 bp stem capped by the pentanucleotide CGAAG to promote intramolecular hairpin formation. The control sequence A10–T10 consists of all native nucleotides. The three FdU-containing sequences differ in the location of FdU nucleotides as shown.
Mentions: The DNA sequences used for these studies were designed to favor intramolecular hairpin formation and to be stable at physiological temperature. Each of the four hairpin DNA sequences consisted of a 10 bp stem consisting of all A–T (or A–FdU) base pairs. The loop region included the GAA sequence that was previously shown to promote hairpin formation (12). The loop was closed by a single C–G base pair. Gel-electrophoresis studies (15% native PAGE gel) demonstrated a single species was present (Supplementary Figure S8) and subsequent 1H NMR studies demonstrated the chemical shift characteristic for the C–G base pair closing the loop in a similar sequence (13). Four DNA hairpins were designed (Figure 2). The sequence ‘A10–T10’ included 10 consecutive T nucleotides at the 5′-terminus and the corresponding 10 consecutive A nucleotides at the 3′-terminus. The ‘5′-FdU’ sequence was identical to the A10–T10 sequence except all of the T nucleotides were substituted by FdU. The ‘3′-FdU’ hairpin DNA sequence had the location of the 10 consecutive A and FdU nucleotides reversed relative to the 5′-FdU hairpin with the 10 consecutive FdU nucleotides located at the 3′-terminus. The ‘Alt-FdU’ sequence had alternating A and FdU nucleotides throughout the stem and thus lacked consecutive FdU nucleotides. These DNA hairpin sequences were designed to evaluate the structural requirements for FdU substitution on formation of complexes with divalent metal ions, including Zn2+ and Mg2+. All DNA sequences were prepared at the University of Calgary DNA Core laboratory and purified by gel-filtration chromatography.Figure 2.

Bottom Line: Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect.DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+).A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Program in Molecular Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

ABSTRACT
We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

Show MeSH
Related in: MedlinePlus